We studied regulation of the Optimedin gene. The expression pattern of the Optimedin A variant in the eye and brain overlaps with that for Pax6 which encodes a protein containing the paired and homeobox DNA-binding domains. The Pax6 gene plays a critical role for the development of eyes, central nervous system and endocrine glands. The proximal promoter of the Optimedin A variant contains a putative Pax6 binding site in the position -86/-70. Pax6 binds this site through the paired domain in vitro as judged by electrophoretic mobility shift assay. Mutations in this site eliminate Pax6 binding as well as stimulation of the Optimedin promoter activity by Pax6 in transfection experiments. Pax6 occupies the binding site in the proximal promoter in vivo as demonstrated by chromatin immunoprecipitation assay. Altogether these results identify the Optimedin gene as a downstream target regulated by Pax6. Although the function of optimedin is still not clear, it is suggested to be involved in cell-cell adhesion and cell attachment to the extracellular matrix. Pax6 regulation of Optimedin in the eye and brain may directly affect multiple developmental processes including cell migration and axon growth.